Project Summary Cell growth is driven by the cell's ability to produce new ribosomes, in which the majority of cellular transcription is devoted to transcribing the ribosomal RNA (rRNA). Cancer cells often rely on aberrant expression of the ribosomal rRNA to increase rates of ribosome assembly in order to stimulate rampant cell growth. Ribosome assembly begins during transcription of the rRNA and the two processes are thought to be co-regulated. However, the relationship between transcription and ribosome assembly is currently unclear, such as the extent of rRNA folding or the composition of ribosomal proteins present during transcription. Moreover, it is unknown how the process of transcription influences rRNA folding and r-protein association. The proposed work aims to develop an in vitro transcription-coupled ribosome assembly system to characterize the effect of transcription on the process of 16S rRNA folding and 30S subunit assembly. The rRNA folding landscape will be examined using a combination of X-ray footprinting and DMS structure probing to provide a map of rRNA structural changes with respect to transcription. Inclusion of ribosomal proteins and assembly factors will elucidate the influence of protein association on the cotranscriptional folding pathway. These experiments will be complemented by single-molecule fluorescence experiments that will characterize kinetic behaviors of individual ribosomal proteins binding to nascent rRNA complexes. The proposed work will provide a novel system for recapitulating in vivo ribosome assembly and generate fundamental insights into transcription-coupled processes. In general, development of these methods will be adaptable for studying different RNPs, such as the snoRNPs or pre-mRNA splicing machinery, as well as studying how cotranscriptional processes are dysregulated in cancer.